*6.1. Trans-Sialidase (TS) Family*

The membrane of parasites as *T. cruzi*, *T. brucei,* or *Trypanosoma rangeli* (*T. rangeli*) is covered by many surface proteins, and most of them are TS or TS-like proteins that are critical for the interactions with the exogenous environment. The TS family is much smaller in *T. brucei* than in *T. cruzi*, and it is absent in *Leishmania major* [42,46,109]. In *T. cruzi*, TS members are localized on the membrane surface of metacyclic, bloodstream trypomastigotes, and intracellular amastigotes and are involved in host-parasite interaction processes [110–113]. They can present a glycosylphosphatidylinositol (GPI) anchor, although this can be removed by the action of a phosphatidylinositol phospholipase C, and then TSs can be released into the bloodstream. TSs are mainly distributed along the flagellum, cell body, and flagellar pocket of the parasite as mucins [114,115].

It is the largest family in *T. cruzi* considering all strains and all their members share the VTVxNVxLYNR motif [116], although some of them present a degeneration of this motif. The first estimates based on the CL Brener genome displayed that the TS family had around 1430 members and 639 pseudogenes [42,112,117] and subsequent studies with strains as Y, Dm28c, TCC or Sylvio X10/1 obtained similar numbers. However, Bug2148 strain displayed 2325 copies [47], almost the double, which could be caused by the hybrid origin of the strain, although the percentage of TS genes with respect to the total of the genetic content is very similar to other strains. Moreover, many TSs are found near the telomeric and sub-telomeric regions, which may cause collapsed assemblies and lead to under or over-representations of the genes. This implies that part of the TS expansion is due to their chromosomal location as we explained before. The other reason is the host immune system pressure to which the TSs are exposed [75] since they are targets of both humoral and cell-mediated immune responses [112].

The best-characterized function of this family is the trans-sialidase catalytic activity, which was first described in 1980 [118]. Posterior studies demonstrated that *T. cruzi* is unable to synthesize their own sialic acids and uses the TSs to incorporate sialic acids from host-cell sialoglycoconjugates to acceptor molecules of their membranes as mucins [119–124]. This sialylation confers a negatively charged coat that protects the trypomastigotes from being killed by human anti-α galactosyl antibodies [125]. A neuraminidase was described in TSs, although it is only active when suitable Gal acceptors are present. It was suggested that this neuraminidase activity just represents around 5% of the total activity of the TS enzyme [126].

Other studies suggest that the TS activity has a key role during the *T. cruzi* infection for parasite survival and the establishment of an effective infection [127]. TSs can interact with different cells from the mammalian hosts, as thymocytes, CD4 <sup>+</sup> and CD8 <sup>+</sup> T cells, B cells, cardiac fibroblasts, endothelial cells, platelets, neurons, and Schwann cells [117]. However, the critical residues that are necessary for the catalytic activity were identified just in a few genes and other roles related to host-ligand interactions and immune regulation were proposed [110,116]. Therefore, renaming this protein family would be advisable since not all their members have TS activity.

Genes encoding TS or TS-like genes were first classified into four groups according to their sequence similarity and functional properties [112,128,129] (Figure 4). TSs of the group I have trans-sialidase and/or neuraminidase catalytic activities [130] and were described in *T. rangeli* too [131]. Interestingly, *T. rangeli* lacks the trans-sialidase activity, retaining only the sialidase [132]. Some of the group I members in *T. cruzi* were the SAPA (shed acute-phase antigen), TS-epi, and TCNA (*T. cruzi* neuraminidase) proteins [116,129], which have active trans-sialidase and neuraminidase activities and are expressed in trypomastigotes [133] (except TS-epi, which is expressed and active in epimastigotes). Both SAPA and TCNA have an N-terminal catalytic region and a C-terminal extension with a tandem repeat of 12 amino acids (SAPA repeats), which consensus sequence is DSSAH [S/G]TPSTP [A/V], and a GPI anchor [134]. Conversely, TS-epi lacks the SAPA repeats and the GPI anchor [135].

**Figure 4.** First classification of TS members. Four groups were described according to their sequence similarity and functional properties. The structure and functions of each group are displayed as well as the known members with their host and parasite-stage in which they are expressed. BT: bloodstream trypomastigotes; A: amastigotes; MT: metacyclic trypomastigotes; E: epimastigotes.

– – TSs of group II are expressed in trypomastigotes and intracellular amastigotes and were also described in *T. rangeli* [136]. This group comprises members of the so-called GP85 glycoproteins (ASP-1, ASP-2, TSA-1, Tc85, SA85, GP82, and GP90 among others) [137] which are related to host-cell attachment [138–142], strong antibody responses in mice and humans [143–145] and *T. cruzi*

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internalization and invasion [141,146–148]. They shared with the TSs of group I, apart from the common TS motif VTVxNVxLYNR, the motifs known Asp-box (SxDxGxTW) and the C-terminal GPI anchor.

Group III is formed by TSs found in bloodstream trypomastigotes as CRP, FL160, CEA, and Trypomastigote Excreted-Secreted Antigens (TESA), which can inactivate both the classical and the alternative pathways of the complement system protecting the parasite from lysis [149–151]. In addition, the TS group IV have TSs with the characteristic motif VTVxNVxLYNR, but with unknown functions.

However, a study of 2011 with the CL Brener strain established a different classification in eight groups by a sequence cluster analysis [110]. The sequence structure of this classification is displayed in Figure 5. The TSs of each group are defined by specific motifs and show specific activities, being the groups II and V those with more members (around 70% of the TSs in the study). Nevertheless, in databases many members of the TS family are annotated in the *T. cruzi* genomes only as trans-sialidase, without the group they belong to, making more difficult to work with this type of complex sequences.

**Figure 5.** Classification of 2011 of TS members according to a sequence cluster analysis. Each group is defined by specific motifs. Logos of each Asp-box and canonical TS motifs are displayed. Adapted from Freitas, L. M. et al., 2011 [110].

Interestingly, phylogenetic analysis with several species of the *T. cruzi* clade and *T. brucei* showed that the variability of the TS-like sequences seems to be consistent with the aggrupation into eight groups. The detection of each TS group in each specie is displayed in Table 2. Group I TSs were found in all the species, in which two clades were established: the *T. brucei* clade with TSs of *T. brucei*, *T. congolense,* and *T. vivax* among others, and the *T. cruzi* clade with TSs of *T. cruzi*, *T. c. marinkellei* and *T. conorhini* among others [107]. It is important to remember that the TSs of group I are active catalytically, therefore it might be possible that other species just require this type of enzymatic function for their viability, while *T. cruzi* needs more TSs with other functions as interaction with host-cell ligands or immune evasion. Furthermore, other studies revealed that sialidases/sialidase-like proteins similar to all *T. cruzi* TS groups exist in *T. rangeli*, although this parasite exhibits fewer members of the trans-sialidase/sialidase family than *T. cruzi* [152,153]. TSs of group II that belong to different DTU strains were also analyzed in a new phylogenetic tree [113]. The results clustered together strains of the same DTU suggesting that TS group II genes might be used as markers for *T. cruzi* genotyping.


**Table 2.** Presence of at least one member of each TS group in different *Trypanosoma* species.

In this classification of eight groups, SAPA, TCNA, and TS-epi that are active TSs belonging to the previously defined group I, clustered together in the new group I, in which not all the members displayed the catalytic sites. ASP-2, Tc85, SA85, GP82, and GP90, which belonged to the previously defined group II and are related to host-cell attachment and invasion, were also classified in the new group II. In addition, finally, FL160 and other TSs involved in the complement system inhibition of the previous group III, clustered in the new group III too.

Regarding the common motifs in these TS groups, all of them have the canonical TS motif (VTVxNVxLYNR), although some variations exist (Figure 5). In fact, there is a small motif (FLY) inside the sequence that can act as a virulence factor [154], and it is only present in group II above all and group IV suggesting a host-cell attachment role in these groups. The Asp-box, previously described in viral and bacterial sialidases as SxDxGxTW [155], appears in some TSs of groups I, II, IV, V, and VI with some variations from the consensus sequence. Most of these TSs have one or two Asp-box, but a few displayed three. The function of this motif in *T. cruzi* remains unknown although it was hypothesized that TSs with these Asp-box could be more capable of binding carbohydrate molecules. The FRIP motif (with the pattern xRxP), which is located upstream the Asp-boxes and involved in binding the carboxylate group of sialic acid [156], was found in groups I, III, IV, VII, and VIII. This implies that although some TSs of these groups are enzymatically inactive, they still preserve carbohydrate-binding properties that could be important for the interaction with the host-cell [157,158]. Finally, tandem repeats, as SAPA repeats, were only found in groups I and IV. Interestingly, this classification forms three different patterns of motif occurrence. Groups I and IV have the most complex structure with all the previously described motifs, despite a few variations in the tandem repeats and the VTVxNVxLYNR motif. Groups II, V, and VI have only the Asp-box and the VTVxNVxLYNR motifs, and groups III, VII, and VIII contain only the FRIP and the VTVxNVxLYNR motifs.

A recent study evaluated the presence of each group in different *T. cruzi* strain genomes [47]. Considering these results and the new genomes of Dm28c and TCC (Figure 6), the TS group V is

the most expanded, with the only exception of Sylvio X10/1 strain. TS group V was associated with antigenic variation allowing the adaptation of the parasites to the host environment [110]. TS group II is the second most abundant cluster, which contains trans-sialidases with host-interaction functions, and TS group I is the most expanded just in Sylvio X10/1 strain. Interestingly, TS group I that have the enzymatically active trans-sialidases, is much less abundant as predicted among strains. TS group III contains trans-sialidases that inhibit the complement pathways, and the different percentage of these trans-sialidases between strains could explain their different sensibility to the complement lysis. Finally, TS groups IV and VII are the less expanded, being absent in some strains as Sylvio X10/1, B7, or Dm28c. Therefore, this fact, in addition to the distinct distribution of Sylvio X10/1, could be caused by the quality of the assembled genomes and/or the annotation of them, being impossible to discard the presence of those TS groups or a few differences in the percentage of each TS group among strains.

**Figure 6.** TS group distribution in genomes of different strains of *T. cruzi* and B7 strain of *T. cruzi*  **Figure 6.** TS group distribution in genomes of different strains of *T. cruzi* and B7 strain of *T. cruzi marinkellei*. The percentage of each TS group is displayed. BNEL: CL Brener Non-Esmeraldo-like; BEL: CL Brener Esmeraldo-like.
